The role of nutrition in analysis of risk factors and short-term outcomes for late-onset necrotizing enterocolitis among very preterm infants: a nationwide, multicenter study in China.

BACKGROUND: Necrotizing enterocolitis (NEC) is a serious gastrointestinal disease, primarily affects preterm newborns and occurs after 7 days of life (late-onset NEC, LO-NEC). Unfortunately, over the past several decades, not much progress has been made in its treatment or prevention. This study aimed to analyze the risk factors for LO-NEC, and the impact of LO-NEC on short-term outcomes in very preterm infants (VPIs) with a focus on nutrition and different onset times. METHOD: Clinical data of VPIs were retrospectively collected from 28 hospitals in seven different regions of China from September 2019 to December 2020. A total of 2509 enrolled VPIs were divided into 2 groups: the LO-NEC group and non-LO-NEC group. The LO-NEC group was divided into 2 subgroups based on the onset time: LO-NEC occurring between 8 ~ 14d group and LO-NEC occurring after 14d group. Clinical characteristics, nutritional status, and the short-term clinical outcomes were analyzed and compared among these groups. RESULTS: Compared with the non-LO-NEC group, the LO-NEC group had a higher proportion of anemia, blood transfusion, and invasive mechanical ventilation (IMV) treatments before NEC; the LO-NEC group infants had a longer fasting time, required longer duration to achieve the target total caloric intake (110 kcal/kg) and regain birthweight, and showed slower weight growth velocity; the cumulative dose of the medium-chain and long-chain triglyceride (MCT/LCT) emulsion intake in the first week after birth was higher and breastfeeding rate was lower. Additionally, similar results including a higher proportion of IMV, lower breastfeeding rate, more MCT/LCT emulsion intake, slower growth velocity were also found in the LO-NEC group occurring between 8 ~ 14d when compared to the LO-NEC group occurring after 14 d (all (P < 0.05). After adjustment for the confounding factors, high proportion of breastfeeding were identified as protective factors and long fasting time before NEC were identified as risk factors for LO-NEC; early feeding were identified as protective factors and low gestational age, grade III ~ IV neonatal respiratory distress syndrome (NRDS), high accumulation of the MCT/LCT emulsion in the first week were identified as risk factors for LO-NEC occurring between 8 ~ 14d. Logistic regression analysis showed that LO-NEC was a risk factor for late-onset sepsis, parenteral nutrition-associated cholestasis, metabolic bone disease of prematurity, and extrauterine growth retardation. CONCLUSION: Actively preventing premature birth, standardizing the treatment of grade III ~ IV NRDS, and optimizing enteral and parenteral nutrition strategies may help reduce the risk of LO-NEC, especially those occurring between 8 ~ 14d, which may further ameliorate the short-term clinical outcome of VPIs. TRIAL REGISTRATION: ChiCTR1900023418 (26/05/2019).

Sensitive detection of HSP70 using a current-amplified biosensor based on antibody-loaded PS-AuNPs@Cys/Au modified ITO chip.

A biosensing electrochemical platform for heat shock protein 70 (HSP70) has been developed by integrating a three-electrode indium tin oxide (ITO) on a chip. The platform includes modifications to the reference electrode and working electrode for the detection of HSP70. The new platform is constructed by assembly of HSP70 antibody on PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode to create a high HSP70 sensitive surface. The PS-AuNPs@Cys/Au indium tin oxide (ITO) electrode is obtained by immersing the ITO electrode into the PS-AuNPs@Cys solution and performing constant potential deposition at -1.4 V (Ag/AgCl). The PS-AuNPs@Cys/Au film deposited on ITO glass provides a desirable substrate for the immobilization of the HSP70 antibody and improves the loading of antibody between PS-AuNPs@Cys/Au and the electrode resulting in a significant amplification. Under optimal conditions, the fabricated sensor demonstrates a linear range extending from 0.1 ng mL- 1 to 1000 ng mL- 1, with an impressive detection limit of 25.7 pg mL- 1 (S/N = 3). The developed immunoassay method successfully detected the HSP70 content in normal human blood samples and outperformed the ELISA method commonly used for clinical sample analysis.

Impact of organic solvents on the catalytic performance of halohydrin dehalogenase.

Biocatalytic transformations in organic synthesis often require the use of organic solvents to improve substrate solubility and promote the product formation. Halohydrin dehalogenases (HHDHs) are enzymes that catalyze the formation and conversion of epoxides, important synthetic class of compounds that are often sparingly soluble in water and prone to hydrolysis. In this study, the activity, stability, and enantioselectivity of HHDH from Agrobacterium radiobacter AD1 (HheC) in form of cell-free extract were evaluated in various aqueous-organic media. A correlation was discovered between the enzyme activity in the ring-closure reaction and logP of the solvent. Knowledge of such a relationship makes biocatalysis with organic solvents more predictable, which may reduce the need to experiment with a variety of solvents in the future. The results revealed a high enzyme compatibility with hydrophobic solvents (e.g., n-heptane) in terms of activity and stability. Regarding the HHDH applicability in an organic medium, inhibitions by a number of solvents (e.g., THF, toluene, chloroform) proved to be a more challenging problem than the protein stability, especially in the ring-opening reaction, thus suggesting which solvents should be avoided. In addition, solvent tolerance of the thermostable variant ISM-4 was also evaluated, revealing increased stability and to a lesser extent enantioselectivity compared to the wild-type. This is the first time such a systematic analysis has been reported, giving insight into the behavior of HHDHs in nonconventional media and opening new opportunities for the future biocatalytic applications. KEY POINTS: • HheC performs better in the presence of hydrophobic than hydrophilic solvents. • Enzyme activity in the PNSHH ring-closure reaction is a function of the logP. • Thermostability of ISM-4 variant is accompanied by superior solvent tolerance.

Analysis of risk factors for parenteral nutrition-associated cholestasis in preterm infants: a multicenter observational study.

BACKGROUND: It is proposed that the development of parenteral nutrition-associated cholestasis (PNAC) was significantly associated with preterm birth, low birth weight, infection, etc.; however, the etiology and pathogenesis of PNAC are not fully understood. Most of the studies examining PNAC-associated risk factors were single-center studies with relatively small sample sizes. OBJECTIVE: To analyze the risk factors associated with PNAC in preterm infants in China. METHODS: This is a retrospective multicenter observational study. Clinical data on the effect of multiple oil-fat emulsions (soybean oil-medium chain triglycerides-olive oil-fish oil, SMOF) in preterm infants were collected from a prospective multicenter randomized controlled study. A secondary analysis was performed in which preterm infants were divided into the PNAC group and the non-PNAC group based on the PNAC status. RESULTS: A total of 465 cases very preterm infants or very low birth weight infants were included in the study in which 81 cases were assigned to the PNAC group and 384 cases were assigned to the non-PNAC group. The PNAC group had a lower mean gestational age, lower mean birth weight, longer duration of invasive and non-invasive mechanical ventilation, a longer duration oxygen support, and longer hospital stay (P < 0.001 for all). The PNAC group had higher respiratory distress syndrome, hemodynamically significant patent ductus arteriosus, necrotizing enterocolitis (NEC) with stage II or higher, surgically treated NEC, late-onset sepsis, metabolic bone disease, and extrauterine growth retardation (EUGR) compared to the non-PNAC group (P < 0.05 for all). In contrast with the non-PNAC group, the PNAC group received a higher maximum dose of amino acids and fat emulsion, more medium/long-chain fatty emulsion, less SMOF, had a longer duration of parenteral nutrition, lower rates of breastfeeding, higher incidence of feeding intolerance (FI), more accumulated days to achieve total enteral nutrition, less accumulated days of total calories up to standard 110 kcal/kg/day and slower velocity of weight growth (P < 0.05 for all). Logistic regression analysis indicated that the maximum dose of amino acids (OR, 5.352; 95% CI, 2.355 to 12.161), EUGR (OR, 2.396; 95% CI, 1.255 to 4.572), FI (OR, 2.581; 95% CI, 1.395 to 4.775), surgically treated NEC (OR, 11.300; 95% CI, 2.127 ~ 60.035), and longer total hospital stay (OR, 1.030; 95% CI, 1.014 to 1.046) were independent risk factors for the development of PNAC. SMOF (OR, 0.358; 95% CI, 0.193 to 0.663) and breastfeeding (OR, 0.297; 95% CI, 0.157 to 0.559) were protective factors for PNAC. CONCLUSIONS: PNAC can be reduced by optimizing the management of enteral and parenteral nutrition and reducing gastrointestinal comorbidities in preterm infants.

Biocatalytic approach to chiral fluoroaromatic scaffolds.

Ten different fluorinated aromatic epoxides have been tested as potential substrates for halohydrin dehalogenase (HHDH) HheC. The majority of investigated epoxides are useful building blocks in synthetic chemistry applications, with a number of them being polysubstituted. Moderate to high enantioselectivities (ER = 15 → 200) were observed in azidolysis, allowing the synthesis of enantioenriched (R)-azido alcohols containing fluorine in the molecule. In the case where a reaction runs over 50% conversion, enantiopure (S)-epoxides are also available. While o-F-styrene oxide was easily converted into a product, a sterically challenging o-CF3-derivative was not accepted by HheC. In silico probing of the binding site indicates that, in order to accommodate an o-CF3-derivative in the HheC active site, it is necessary to eliminate steric hindrance. Hence, we extended our research by probing several available HheC variants containing relevant modifications in the active site. The active mutant P84V/F86P/T134A/N176A (named HheC-M4) was identified, showing not only high activity towards o-CF3-styrene oxide, but also inverted enantioselectivity (ES = 27). Since (S)-enantioselective HHDHs are rare and therefore valuable for their synthetic application, this enzyme was screened on the initial panel of substrates. The observed (S)-enantioselectivity (ES = 1-111) is ascribed to the formation of the additional space by introduced mutations in HheC-M4, which is also confirmed by classical MD simulations. Successive molecular docking demonstrated that this newly formed tunnel located close to the protein surface is a critical feature of HheC-M4, representing a novel binding site.

Overexpression and kinetic analysis of Ideonella sakaiensis PETase for polyethylene terephthalate (PET) degradation.

Ideonella sakaiensis PET hydrolase (IsPETase) is a well-characterized enzyme for effective PET biodegradation. However, the low soluble expression level of the enzyme hampers its practical implementation in the biodegradation of PET. Herein, the expression of IsPETaseMut, one of the most active mutants of IsPETase obtained so far, was systematically explored in E. coli by adopting a series of strategies. A notable improvement of soluble IsPETaseMut was observed by using chaperon co-expression and fusion expression systems. Under the optimized conditions, GroEL/ES co-expression system yielded 75 ± 3.4 mg·L-1 purified soluble IsPETaseMut (GroEL/ES), and NusA fusion expression system yielded 80 ± 3.7 mg·L-1 purified soluble NusA-IsPETaseMut, which are 12.5- and 4.6-fold, respectively, higher than its commonly expression in E. coli. The two purified enzymes were further characterized. The results showed that IsPETaseMut (GroEL/ES) displayed the same catalytic behavior as IsPETaseMut, while the fusion of NusA conferred new enzymatic properties to IsPETaseMut. Although NusA-IsPETaseMut displayed a lower initial hydrolysis capacity than IsPETaseMut, it showed a 1.4-fold higher adsorption constant toward PET. Moreover, the product inhibition effect of terephthalic acid (TPA) on IsPETase was reduced with NusA-IsPETaseMut. Taken together, the latter two catalytic properties of NusA-IsPETaseMut are more likely to contribute to the enhanced product release by NusA-IsPETaseMut PET degradation for two weeks.

Correlation between Left Atrial Thrombosis and Neutrophil-to-Lymphocyte Ratio upon Non-Valvular Atrial Fibrillation.

BACKGROUND: To explore the correlation between left atrial thrombosis and peripheral blood neutrophil-to-lymphocyte ratio (NLR) in patients with non-valvular atrial fibrillation (NVAF). METHODS: A total of 207 NVAF patients treated from March 2016 to August 2020 were divided into low and high NLR groups. Their clinical data were compared, and the correlations of NLR with clinical indicators and left atrial thrombosis were analyzed. The influencing factors for left atrial thrombosis and their predictive values were investigated. RESULTS: CHADS2 score, CHA2DS2-VASc score, NLR, D-dimer (D-D), serum uric acid (SUA), brain natriuretic peptide (BNP), C-reactive protein (CRP), left atrial dimension (LAD) and ratio of early mitral diastolic flow peak value to early diastolic mitral annular velocity (E/Em) increased, while left atrial ejection fraction (LAEF) decreased in high NLR group compared with those in low NLR group (p < 0.05). NLR was significantly positively correlated with the CHADS2 score, CHA2DS2-VASc score, D-D, SUA, BNP, CRP, LAD and E/Em, and significantly negatively correlated with LAEF (p < 0.0001). Left atrial thrombosis had positive correlations with CHADS2 score, CHA2DS2-VASc score, NLR, D-D, SUA, BNP, CRP, LAD and E/Em, and negative correlation with LAEF (p = 0.000). CHA2DS2-VASc score, NLR, D-D and LAD were risk factors for left atrial thrombosis (p < 0.05). The optimal cut-off value was 1.85 when the area under the curve of NLR for predicting left atrial thrombosis in the case of NVAF was 0.806 [95% confidence interval (CI): 0.746 - 0.865], the sensitivity was 74.82% (95% CI: 0.668 -0.818) and the specificity was 67.65% (95% CI: 0.552 - 0.785). CONCLUSIONS: The increase of NLR prominently elevates the risk of left atrial thrombosis in NVAF patients, and NLR was an independent risk factor, with certain predictive value.

A Statistical Analysis of the Sequence and Structure of Thermophilic and Non-Thermophilic Proteins.

Thermophilic proteins have various practical applications in theoretical research and in industry. In recent years, the demand for thermophilic proteins on an industrial scale has been increasing; therefore, the engineering of thermophilic proteins has become a hot direction in the field of protein engineering. However, the exact mechanism of thermostability of proteins is not yet known, for engineering thermophilic proteins knowing the basis of thermostability is necessary. In order to understand the basis of the thermostability in proteins, we have made a statistical analysis of the sequences, secondary structures, hydrogen bonds, salt bridges, DHA (Donor-Hydrogen-Accepter) angles, and bond lengths of ten pairs of thermophilic proteins and their non-thermophilic orthologous. Our findings suggest that polar amino acids contribute to thermostability in proteins by forming hydrogen bonds and salt bridges which provide resistance against protein denaturation. Short bond length and a wider DHA angle provide greater bond stability in thermophilic proteins. Moreover, the increased frequency of aromatic amino acids in thermophilic proteins contributes to thermal stability by forming more aromatic interactions. Additionally, the coil, helix, and loop in the secondary structure also contribute to thermostability.

Epidemiology and antimicrobial susceptibility of invasive Escherichia coli infection in neonates from 2012 to 2019 in Xiamen, China.

BACKGROUND: Escherichia coli (E. coli) is one of the important causative pathogens of neonatal invasive infection. The epidemiological and clinical profile of invasive E. coli infection in Chinese newborns is not well characterized. METHODS: Ninety-four infants with invasive E. coli infection were categorized into E. coli early onset disease (EOD) group (onset ≤72 h after birth) (n = 46) and E. coli late onset disease (LOD) group (onset > 72 h) (n = 48). We compared and analyzed the clinical characteristics and drug sensitivity profile of early-onset and late-onset E. coli invasive infection in neonates. RESULTS: The incidence of E. coli-EOD and E.coli-LOD was 0.45/1000 live births (LBs) and 0.47/1000 LBs, respectively. The incidence of gestational diabetes mellitus, perinatal fever, urinary tract infection, chorioamnionitis, and positive E. coli culture among mothers in the E. coli-EOD group were significantly higher than that in E. coli-LOD group. The incidence of premature birth, low-birth-weight, nosocomial infection, and hospitalization time were significantly higher in the E. coli-LOD group. The main disease in E. coli-EOD group was pneumonia (main clinical manifestation: dyspnea). The main disease in E. coli-LOD group was sepsis (main clinical manifestation: fever). The sensitivity rates of E. coli strains to ampicillin and piperacillin were low (25.00-28.79%); sensitivity to cephalosporins was also low except ceftazidime (lowest sensitivity rate: 57.14%). Sensitivity to compound preparations containing ß-lactamase inhibitors was high, even for extended spectrum ß-lactamase-positive strains (nearly 100%). CONCLUSION: E. coli is an important cause of invasive infection of newborns in Xiamen, China. E. coli-EOD was largely attributable to perinatal factors, while E. coli-LOD was largely related to nosocomial infection. Compound preparations containing ß-lactamase inhibitor or carbapenem antibiotics should be preferred for neonatal invasive infection by E. coli.

[The Role of Nrf2 Pathway Activation in Hippocampal Neuron Injury of Neonatal Rats with Bilirubin Encephalopathy].

OBJECTIVE: To explore the effect of nuclear factor-erythroid 2-related factor (Nrf2) pathway activation on hippocampal neuron damage in neonatal rats with bilirubin encephalopathy. METHODS: Neonatal rats were randomly assigned to a control group (Control), a model group (Model) and an Nrf2 activator TBHQ (tert-Butylhydroquinone) group (TBHQ), with 20 rats in each group. Bilirubin solution was injected through the cerebellomedullary cistern to establish the neonatal rat model of bilirubin encephalopathy. Neurobehavioral changes were observed in rats and the water content of the brain tissue was measured. Nissl staining was done to observe the damage of hippocampal neurons. TUNEL staining was used to observe the apoptosis of hippocampal neurons. Colorimetric analysis was done to determine the Caspase-3 activity in the hippocampus. The content of malondialdehyde (MDA) and reduced glutathione (GSH) and the activity of superoxide dismutase (SOD) in the hippocampus were examined by chemical analysis. qRT-PCR and Western blot were done to measure the expression of Nrf2 and heme oxygenase-l (HO-1) mRNA and proteins in the hippocampus. RESULTS: After injection of bilirubin into the cerebellomedullary cistern, the young rats in the Model group and the TBHQ group showed different degrees of neurological abnormalities, while those in the control group showed no significant neurobehavioral abnormalities. Compared with the Control group, the Model group had severe neuronal damage, and the water content of brain tissue, the apoptosis of hippocampal neurons, the activity of Caspase-3 and the content of MDA content significantly increased ( P<0.01), while the SOD activity, GSH content, the expression of Nrf2 and HO-1 mRNA and proteins significantly decreased ( P<0.05). Compared with the Model group, neuronal damage was improved in the TBHQ group, and the water content of brain tissue, apoptosis of hippocampal neurons, activity of Caspase-3 and MDA content were all significantly reduced ( P<0.01), while SOD activity, GSH content and the expression of Nrf2 and HO-1 mRNA and proteins were significantly increased ( P<0.05). CONCLUSION: Activation of the Nrf2 pathway can improve hippocampal neuronal damage in neonatal rats with bilirubin encephalopathy and inhibit neuronal apoptosis and the oxidation reaction.

[Clinical effect of an additional maintenance dose of caffeine before ventilator weaning in preterm infants with respiratory distress syndrome: a prospective randomized controlled trial].

OBJECTIVE: To study the clinical effect of an additional maintenance dose (5 mg/kg) of caffeine citrate injection at 1 hour before ventilator weaning in improving the success rate of ventilator weaning in preterm infants (gestational age ≤32 weeks) with respiratory distress syndrome (RDS) on mechanical ventilation. METHODS: A total of 338 preterm infants with RDS (gestational age of ≤32 weeks) who were admitted to the Neonatal Intensive Care Unit of Xiamen Maternal and Child Health Hospital from January 2017 to December 2019 and treated with mechanical ventilation were enrolled. They were randomly divided into an observation group and a routine group, with 169 infants in each group. Both groups received early routine treatment with caffeine. The infants in the observation group received an additional maintenance dose of caffeine citrate injection at 1 hour before ventilator weaning. The two groups were compared in terms of reintubation rate and number of apnea episodes within 48 hours after ventilator weaning, changes in blood gas parameters, blood glucose, heart rate, and mean blood pressure at 2 hours after ventilator weaning, and incidence rates of major complications during hospitalization. RESULTS: Compared with the routine group, the observation group had significantly lower reintubation rate (P=0.034) and number of apnea episodes (≥2 times/day) (P=0.015) within 48 hours after ventilator weaning. Compared with the routine group at 2 hours after ventilator weaning, the observation group had a significantly higher pH value and a significantly lower arterial partial pressure of carbon dioxide (P < 0.05), while there were no significant differences between the two groups in arterial partial pressure of oxygen, blood glucose, heart rate, and mean blood pressure (P > 0.05). During hospitalization, the observation group had a significantly lower incidence rate of intraventricular hemorrhage than the routine group (P=0.048), but there were no significant differences between the two groups in the incidence rates of bronchopulmonary dysplasia, necrotizing enterocolitis, retinopathy of prematurity, and periventricular leukomalacia and mortality rate (P > 0.05). CONCLUSIONS: An additional maintenance dose of caffeine citrate injection at 1 hour before ventilator weaning is safe and effective in improving the success rate of ventilator weaning in preterm infants with RDS and thus holds promise for clinical application.

[Safety of two ventilator weaning strategies after high-frequency oscillatory ventilation in preterm infants with respiratory distress syndrome: a prospective randomized controlled trial].

OBJECTIVE: To study the safety of two ventilator weaning strategies after high-frequency oscillatory ventilation (HFOV) for the treatment of neonatal respiratory distress syndrome (NRDS) in preterm infants. METHODS: A prospective randomized controlled trial was conducted for 101 preterm infants with NRDS, with a gestational age of ≤32+6 weeks or a birth weight of ≤1 500 g, who were admitted to the neonatal intensive care unit of Xiamen Maternal and Child Health Hospital from January 1, 2019 to June 30, 2020. The infants underwent HFOV as the preferred treatment. The infants were randomly divided into an observation group (50 infants with direct weaning from HFOV) and a control group (51 infants with weaning after HFOV was switched to conventional mechanical ventilation). The two groups were compared in terms of failure rate of ventilator weaning within 72 hours, changes in blood gas parameters at 2 hours before weaning and at 2 and 24 hours after weaning, respiratory support therapy, incidence rates of complications, and outcome at discharge. RESULTS: There was no significant difference in the failure rate of ventilator weaning within 72 hours (8% vs 14%, P > 0.05). The observation group had a significantly shorter duration of mechanical ventilation than the control group [(64±39) hours vs (88±69) hours, P < 0.05]. There were no significant differences between the two groups in the duration of mechanical ventilation, total oxygen supply time, blood gas parameters before and after ventilator weaning, incidence rates of complications, and outcome at discharge (P > 0.05). CONCLUSIONS: For preterm infants with NRDS, the strategy of weaning directly from HFOV is safe and reliable and can reduce the duration of invasive mechanical ventilation, and therefore, it holds promise for clinical application.

Enzyme immobilization on glass fiber membrane for detection of halogenated compounds.

Enzyme immobilization using inorganic membranes has enticed increased attention as they not only improve enzyme stability, but also furnish user-friendly biodevices that can be tailored to different applications. Herein, we explored the suitability of the glass fiber membrane for enzyme immobilization and its application for halocarbon detection. For this, halohydrin dehalogenase (HheC) and bovine serum albumin were crosslinked and immobilized on a glass fiber membrane without membrane functionalization. Immobilized HheC exhibited higher storage stability than its free counterpart over 60 days at 4 °C (67% immobilized vs. 8.1% free) and 30 °C (77% immobilized vs. 57% free). Similarly, the thermal endurance of the immobilized HheC was significantly improved. The practical utility of the membrane-immobilized enzyme was demonstrated by colorimetric detection of 1,3-dichloro-2-propanol (1,3-DCP) and 2,3-dibromo-1-propanol (2,3-DBP) as model analytes. Under optimized conditions, the detection limits of 0.06 mM and 0.09 mM were achieved for 1,3-DCP and 2,3-DBP, respectively. The satisfactory recoveries were observed with spiked river and lake water samples, which demonstrate the application potential of immobilized HheC for screening contaminants in water samples. Our results revealed that the proposed frugal and facile approach could be useful for enzyme stabilization, and mitigation of halocarbon pollution.

Osteoprotegerin/bone morphogenetic protein 2 combining with collagen sponges on tendon-bone healing in rabbits.

INTRODUCTION: The aim was to investigate the effect of collagen sponges (CS) as a delivery device for osteoprotegerin (OPG)/bone morphogenetic protein 2 (BMP-2) and support matrix on the tendon-bone healing after anterior crusicate ligament (ACL) reconstruction in modeled rabbits. MATERIALS AND METHODS: Sixty New Zealand white rabbits were randomly divided into four groups based on treatments they received at the tendon-bone interface after left knee ACL reconstruction: the control group, OPG/BMP-2, CS, and OPG/BMP-2/CS combination. At 4, 8 and 12 weeks post-surgery, five rabbits from each group were euthanized to examine the tendon-bone healing. Levels of OPG and BMP-2 in synovial fluid, the bone tunnel enlargement value, the histomorphological typing of tendon-bone interface, and the bone tunnel area of the tendon-bone interface were compared among different treatments. RESULTS: The OPG/BMP-2/CS combination treatment group had the highest levels of OPG and BMP-2 in synovial fluid (both P < 0.05), the greatest number of Sharpey-like collagen fibers at all test points (P < 0.05), the most fibrocartilage enthesis on week 12, the greatest bone tunnel area (P < 0.05), and the greatest decrease in bone tunnel enlargement on week 12 (P < 0.05). Histomorphological typing of tendon-bone interface of all groups showed changes varying from tendon-bone separation to firm healing, and the change was most significant in the OPG/BMP-2/CS combination treatment group. CONCLUSION: CS treatment alone serves as a fixing support, and CS combining with growth factors OPG/BMP-2 ensures slow and stable release of OPG/BMP-2, significantly improves the tendon-bone healing in the rabbit ACL model.

A paper-based whole-cell screening assay for directed evolution-driven enzyme engineering.

Directed evolution has become an important method to unleash the latent potential of enzymes to make them uniquely suited for human purposes. However, the need for a large reagent volume and sophisticated instrumentation hampers its broad implementation. In an attempt to address this problem, here we report a paper-based high-throughput screening approach that should find broad application in generating desired enzymes. As an example case, the dehalogenation reaction of the halohydrin dehalogenase was adopted for assay development. In addition to visual detection, quantitative measurements were performed by measuring the color intensity of an image that was photographed by a smartphone and processed using ImageJ free software. The proposed method was first validated using a gold standard method and then applied to mutagenesis library screening with reduced consumption of reagents (i.e., ≤ 10 µl per assay) and a shorter assay time. We identified two active mutants (P135A and G137A) with improved activities toward four tested substrates. The assay not only consumes less reagents but also eliminates the need for expensive instrumentation. The proposed method demonstrates the potential of paper-based whole-cell screening coupled with digital image colorimetry as a promising approach for the discovery of industrially important enzymes.Key Points• A frugal method was developed for directed enzyme evolution.• Mutagenesis libraries were successfully screened on a paper platform.• Smartphone imaging was efficiently used to measure enzyme activities.

Enzyme-based detection of epoxides using colorimetric assay integrated with smartphone imaging.

Epoxides are widely used chemicals, the determination of which is of paramount importance. Herein, we present an enzyme-based approach for noninstrumental detection of epoxides in standard solution and environmental samples. Halohydrin dehalogenase (HheC) as a biological recognition element and epichlorohydrin as a model analyte were evaluated for sensing. The detection is based on the color change of the pH indicator dye bromothymol blue caused by the HheC-catalyzed ring-opening of the epoxide substrate. The color change is then exploited for the determination of epoxide using a smartphone as an image acquisition and data processing device, eliminating the need for computer-based image analysis software. The color parameters were systematically evaluated to determine the optimum quantitative analytical parameter. Under optimal conditions, the proposed enzyme-based detection system showed a linear range of 0.13-2 mM with a detection limit of 0.07 mM and an assay time of 8 Min. In addition, the repeatability expressed as relative standard deviation was found to be below 5% (n = 6). Validation with gas chromatographic analyses showed that the proposed enzyme-based epoxide detection could be an alternative way in the quantitative determination of epoxides, and particularly useful in resource-limited settings.

Association of genetic polymorphisms of miR-145 gene with telomere length in omethoate-exposed workers.

Omethoate, an organophosphorous pesticide, causes a variety of health effects, especially the damage of chromosome DNA. The aim of the study was to assess the correlation between polymorphisms of encoding miRNA genes and telomere length in omethoate-exposure workers. 180 workers with more than 8 years omethoate-exposure and 115 healthy controls were recruited in the study. Genotyping for the selected single nucleotide polymorphisms loci were performed using the flight mass spectrometry. Real-time fluorescent quantitative polymerase chain reaction(PCR) method was applied to determine the relative telomere length(RTL) in human peripheral blood leukocytes DNA. After adjusting the covariate of affecting RTL, covariance analysis showed that the female was significantly longer than that of the male in control group(P < 0.046). For the miR-145 rs353291 locus, this study showed that RTL of mutation homozygous AG+GG individuals was longer than that of wild homozygous AA in the exposure group (P = 0.039). In the control group, RTL with wild homozygous TT genotype in miR-30a rs2222722 polymorphism locus was longer than that of the mutation homozygous CC genotype (P = 0.038). After multiple linear regression analysis, the independent variables of entering into the model were omethoate-exposure (b = 0.562, P < 0.001), miR-145 rs353291 (AG+GG) (b = 0.205, P = 0.010). The prolongation of relative telomere length in omethoate exposed workers was associated with AG+GG genotypes in rs353291 polymorphism of encoding miR-145 gene.

Protective effects of quercetin against inflammation and oxidative stress in a rabbit model of knee osteoarthritis.

Hit, Lead & Candidate Discovery This study investigated the effects of a natural phenolic compound quercetin on surgical-induced osteoarthritis (OA) in rabbits. Forty-eight New Zealand White rabbits were used to establish OA model by Hulth modified method, and subsequently randomized into untreated OA group (treatment was drinking water), celecoxib treated group (celecoxib 100 mg kg-1 by gavage), and quercetin treated group (25 mg kg-1 by gavage). Sixteen nonoperated rabbits served as the normal controls (drinking water was given). The treatment (length: 4 weeks) started on the 5th week postoperation when the OA pathological changes were manifested. Expressions of superoxide dismutase (SOD), matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in serum, synovial fluid, and synovial tissue were measured at 8 and 12 weeks postoperation. Pathological analysis was performed with synovial tissue section and Osteoarthritis Research Society International histopathology grading and staging scores were determined. The quercetin treated group showed higher SOD and TIMP-1 expressions but lower MMP-13 expression than untreated OA group in the serum, synovial fluid and synovial tissues (p < .05). There was no significant difference in the SOD, MMP-13 and TIMP-1 expressions between the quercetin-treated and celecoxib-treated groups. The MMP-13/TIMP-1 ratio of the quercetin treated group was significantly lower than that of the untreated OA group (p < .05). Quercetin can up-regulate SOD and TIMP-1, down-regulate MMP-13, and improve the degeneration of OA through weakening the oxidative stress responses and inhibiting the degradation of cartilage extracellular matrix.

Telomere length in workers was effected by omethoate exposure and interaction between smoking and p21 polymorphisms.

Omethoate is an organophosphorus pesticide that poses a major health hazard, especially DNA damage. The purpose of this study was to investigate the factors affecting telomere length in workers exposed to omethoate by analyzing the interaction between cell cycle gene polymorphism and environmental factors. The exposure group consisted of 118 workers exposed to omethoate for 8-10 years, the control group comprised 115 healthy people without occupational toxicant exposure history. The telomere length of genomic DNA from peripheral blood leucocyte was determined with real-time PCR. Polymerase chain reaction and restriction fragment length polymorphism was used to detect the polymorphisms in p53, p21 and MDM2 gene. The telomere length in the (CA + AA) genotypes for p21 rs1801270 polymorphism was longer than that in the CC genotype in control group (P = 0.015). The generalized linear model analysis indicated the interaction of the p21 rs1801270 polymorphic (CA + AA) genotypes and smoking has a significant effect on telomere length (ß = -0.258, P = 0.085). The prolongation of telomere length in omethoate-exposed workers was associated with genotypes (CA + AA) of p21 rs1801270, and interactions of (CA + AA) genotypes and smoking factor.